Volume 131, number 2 FEBS LETTERS August 1981 CALCIUM AND CALMODULIN-DEPENDENT PROTEIN PHOSPHORYLATION IN RABBIT ILEUM Leslie TAYLOR, Victoria J. GUERINA, Mark DONOWITZ, Michael COHEN and Geoffrey W. G. SHARP* Departments of Physiology and Medicine, Tufts University School of Medicine and The New England Medical Center Hospital Boston, MA 02111, USA Received 13 July 1981 1. Introduction Evidence is accumulating to suggest that calcium is a physiological regulator of intestinal electrolyte transport [ 1-3]. Conditions which increase intracel- lular calcium such as the use of the calcium ionophore A23187 [ 1,2] or exposure to neurohumoral sub- stances such as serotonin [4] or carbachol [ 1] cause stimulation of intestinal chloride secretion and/or inhibition ofNa ÷and C1- absorption. To the contrary, conditions which decrease intracellular calcium, such as exposure to the calcium channel blocker verapamil, stimulate Na ÷ and C1- absorption [3]. Calmodulin may be involved in these effects since the anti-psy- chotic drug trifluoperazine (an inhibitor of the cal- cium-calmodulin complex) inhibited intestinal secre- tion caused by the calcium ionophore A23187 [5,6]. The intracellular mechanisms by which calcium and calmodulin affect intestinal ion transport are not known. However, as calcium and calmodulin affect phosphorylation and function of specific proteins in several other systems [7,8], phosphorylation is a potential control mechanism for ileal electrolyte transport. These studies are the first demonstration that calcium and calmodulin can cause phosphoryla- tion of intestinal peptides. 2. Materials and methods Fed white male New Zealand rabbits (2-2.5 kg * Present address: Department of Pharmacology,New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Address correspondence and reprint requests to: Dr Mark Donowitz, GastroenterologyUnit, Tufts-New England Medi- cal Center, 171 Harrison Avenue, Boston, MA 02111, USA body wt) were anesthetized with sodium pentobarbital and the distal ileum removed. After washing with cold Ringer's-HCO3 solution, mucosal scrapings were obtained on ice using glass slides. This technique has been demonstrated to obtain primarily villous tip cells and some crypt cells [9]. 150 mg tissue was homogenized in 7.5 ml 5 mM MgC12, 10% sucrose, 50 mM Tris-HC1 (pH 7.5) using a ground-glass hand homogenizer. The homogenate, containing ~200/2g protein, was preincubated in a 100/21 reaction mix- ture containing 1 mM EGTA, 5 mM MgC12, 10% sucrose, 50 mM Tris-HC1 (pH 7.5) and, where indi- cated, 1.1 mM CaC12, and different concentrations of calmodulin and trifluoperazine, for 1 min at 37°C. The phosphorylation reaction was started by the addition of [32p] ATP at a final concentration of 5/2M. The reaction was stopped after 20 s by the addition of 50/21 of a solution containing 0.1 mM EDTA, 5% SDS, 200 mM dithiothreitol and 50 mg/ml pyronin Y followed immediately by immersion in a boiling water bath for 2 min. The mixture was then placed in a 37°C water bath for 20 rain. Samples, contain- ing --40/2g protein, were subjected to electrophoresis on a polyacrylamide continuous gradient slab gel (5-15% acrylamide) according to [10]. The gels were subjected to autoradiography using XAR-5 Kodak film (Eastman Kodak, Rochester NY) with exposure times ranging from 4-48 h. The autoradio- graphs were subsequently analyzed by use of a Zeneih scanning densitometer (Biomed Products, Chicago IL). Amount of phosphorylation was assumed propor- tional to peak height of the densitometry scan [ 11 ]. Between 6 and 8 experiments were performed for each experimental condition. [TP2P] Adenosine triphosphate (spec. act. 3-10 Ci/ mmol) was obtained from New England Nuclear Published by Elsevier/North-Holland Biomedical Press 322 00145793/81/0000-0000/$02.50 © 1981 Federation of European BiochemicalSocieties